Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
  • C07K17/02—Peptides being immobilised on, or in, an organic carrier
  • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
  • B01D15/3804—Affinity chromatography
  • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G or L chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/282—Porous sorbents
  • B01J20/285—Porous sorbents based on polymers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
  • B01J20/289—Phases chemically bonded to a substrate, e.g. to silica or to polymers bonded via a spacer
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
  • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
  • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
  • B01J20/3268—Macromolecular compounds
  • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
  • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
  • C07K17/02—Peptides being immobilised on, or in, an organic carrier
  • C07K17/10—Peptides being immobilised on, or in, an organic carrier the carrier being a carbohydrate
  • C07K17/12—Cellulose or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/515—Complete light chain, i.e. VL + CL
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Definitions

• the present invention relates to the field of separation of biomolecules. More specifically, it relates to a separation matrix for affinity chromatography and separation of biomolecules based on the presence of a kappa light chain, such as immunoglobulins and immunoglobulin fractions. The invention also relates to methods of using said separation matrix.
• Immunoglobulins and immunoglobulin fragments represent the most prevalent biopharmaceutical products in either manufacture or development worldwide.
• the high commercial demand for, and hence value of, this particular therapeutic market has led to the emphasis being placed on pharmaceutical companies to maximize the productivity of their respective manufacturing processes whilst controlling the associated costs.
• Affinity chromatography typically on matrices comprising staphylococcal Protein A or variants thereof, is normally used as one of the key steps in the purification of intact immunoglobulin molecules.
• the highly selective binding of Protein A to the Fc chain of immunoglobulins provides for a generic step with very high clearance of impurities and contaminants.
• matrices comprising Protein L derived from Finegoldia magna (formerly Peptostreptococcus Magnus) (B Akerstrbm, L Bjbrck: J. Biol. Chem. 264, 19740-19746, 1989; W Kastem et al: J. Biol. Chem. 267, 12820-12825, 1992; B HK Nilson et al: J. Biol. Chem. 267, 2234-2239, 1992 and US Pat. 6,822,075) show great promise as a purification platform providing the high selectivity needed.
• Protein L matrices are commercially available as for instance CaptoTM L from CytivaTM and can be used for separation of kappa light chain-containing proteins such as intact antibodies, Fab fragments, scFv fragments, domain antibodies etc. About 75% of the antibodies produced by healthy humans have a kappa light chain and about 90% of therapeutic monoclonal antibodies and antibody fragments contain kappa light chains (Carter, P., Lazar, G. Next generation antibody drugs: pursuit of the 'high-hanging fruit'. Nat Rev Drug Discov 17, 197-223 (2018). https://doi.org/10.1038/nrd.2017.227). Any bioprocess chromatography application requires comprehensive attention to definite removal of impurities and/or contaminants.
• Such impurities and/or contaminants can for example be non-eluted molecules adsorbed to the stationary phase or matrix in a chromatographic procedure, such as nondesired biomolecules or microorganisms, including for example proteins, carbohydrates, lipids, bacteria and viruses.
• the removal of such impurities and/or contaminants from the matrix is usually performed after a first elution of the desired product in order to regenerate the matrix before subsequent use.
• Such removal usually involves a procedure known as cleaning-in-place (Cl P), wherein agents capable of either inactivating or eluting impurities from the stationary phase are used.
• One such class of agents often used with chromatography media is alkaline solutions that are passed over the matrix.
• the most extensively used cleaning and sanitizing agent is NaOH, and it is desirable to use it in concentrations ranging from 0.05 up to e.g. 1 M, depending on the degree and nature of contamination and impurity.
• Protein L is however a rather alkali-sensitive protein compared to e.g. Protein A and only tolerates up to about 15 mM NaOH over a large number of cycles. This means that additional, less desirable cleaning solutions, e.g. urea or guanidinium salts, may also have to be used in order to ensure sufficient cleaning.
• the present inventors have attained to solve the above-mentioned problem by providing a separation matrix comprising kappa light chain-binding ligands covalently coupled to a porous support, wherein said kappa light chain-binding ligands comprise, consists essentially of, or consists of multimers of alkali- stabilized Finegoldia magna (formerly Peptostreptococcus Magnus) Protein L domains; and said porous support is a convection-based chromatography matrix.
• the convection-based chromatography matrix may be a fibrous substrate.
• Said fibrous substrate may be based on electrospun polymeric fibers or cellulose fibers, optionally non-woven fibers.
• the polymer may be selected from the group consisting of cellulose, cellulose acetate, polysulfones, polyamides, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polyethylene oxide, and mixtures thereof.
• the fibrous substrate is a fibrous non-woven polymer matrix.
• the fibers comprised in said fibrous substrate may have a cross-sectional diameter of 10-1000 nm, such as 200-800 nm, 200-400 nm or 300-400 nm.
• the ligands may be bound to divinyl sulfone functional groups coupled to glycidol groups grafted onto the fibrous substrate.
• Said kappa light chain-binding ligands may comprise at least two alkali-stabilized Protein L domains.
• the alkali-stabilized Protein L domains may be selected from the group comprising of functional variants of a Bl domain, a B2 domain, a B3 domain, a B4 domain, a B5 domain, a C2 domain, a C3 domain, a C4 domain and a DI domain of Finegoldia magna (formerly Peptostreptococcus Magnus) Protein L, wherein the positions which in an alignment corresponds to positions 10 and 45 in a B2 domain (SEQ ID NO 1) are histidine, and the position which in an alignment corresponds to position 60 in a B2 domain (SEQ ID NO 1) is a tyrosine or a glutamine.
• the alkali-stabilized Protein L domains are chosen from the group comprising a B2 domain, a B3 domain, a B4 domain, a C2 domain, a C3 domain,
• the alkali-stabilized Protein L domains may have at least 90%, 95% or 98% sequence identity or a 77,5 % sequence similarity as determined by BLOSUM matrix of 75, with a gap open penalty of 12, a gap extension penalty of 3, with any one of the amino acid sequences SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18 or SEQ ID NO 19, wherein the positions which in an alignment corresponds to positions 10 and 45 in SEQ ID NO 1, and the position which in an alignment corresponds to position 60 in SEQ ID NO 1 are not variable.
• the alkali-stabilized Protein L domains may have at least 90%, 95% or 98% sequence identity, or a 77.5% sequence similarity as determined by BLOSUM matrix of 75, with a gap open penalty of 12, a gap extension penalty of 3, with any one of the amino acid sequences SEQ ID NQ:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NQ:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:37.
• the ligand density for the separation matrix may be at least 20 mg/ml porous support, or at least 25 mg/ml porous support, or at least 30 mg/ml porous support, or at least 35 mg/ml porous support, or at least 40 mg/ml porous support, or at least 45 mg/ml porous support, or at least 50 mg/ml porous support.
• the separation matrix may have a dynamic binding capacity (DBC) of a kappa light chain-comprising antibody, such as Trastuzumab, of 25 g/mL at 10% breakthrough when running at a flow of 10 mL/min in a 0.4 mL HiTrapTM device.
• the separation matrix may have a dynamic binding capacity (DBC) of a kappa light chain-comprising antibody, such as Trastuzumab, of 45 g/mL at 10% breakthrough when running at a flow of 10 mL/min in a 0.4 mL HiTrapTM device.
• a method of isolating a kappa light chain-containing protein comprising the steps of: a) contacting a liquid sample comprising a kappa light chain-containing protein with a separation matrix; b) washing said separation matrix with one or a combination of several washing liquids; c) eluting the kappa light chain-containing protein from the separation matrix with an elution liquid; and d) cleaning the separation matrix with a cleaning liquid; wherein the separation matrix has a dynamic binding capacity (DBC) of a kappa light chain-comprising antibody, such as Trastuzumab, of 25 g/mL at 10% breakthrough when running at a flow of 10 mL/min in a 0.4 mL HiTrapTM device.
• DBC dynamic binding capacity
• a method for separation of isolating a kappa light chain-binding protein from lambda light chain-containing proteins comprising the steps of: a) contacting a liquid sample comprising a kappa light chain-containing protein with a separation matrix; b) washing said separation matrix with one or a combination of several washing liquids; c) eluting the kappa light chain-containing protein from the separation matrix with an elution liquid; and d) cleaning the separation matrix with a cleaning liquid; wherein the separation matrix has a dynamic binding capacity (DBC) of a kappa light chain-comprising antibody, such as Trastuzumab, of 25 g/mL at 10% breakthrough when running at a flow of 10 mL/min in a 0.4 mL HiTrapTM device.
• DBC dynamic binding capacity
• a method for separation of bispecific antibodies from mono-specific antibodies comprising the steps of: a) contacting a liquid sample comprising a bispecific antibody with a separation matrix; b) washing said separation matrix with one or a combination of several washing liquids; c) eluting the bispecific antibody from the separation matrix with an elution liquid; and d) cleaning the separation matrix with a cleaning liquid; wherein the separation matrix has a dynamic binding capacity (DBC) of a kappa light chain-comprising antibody, such as Trastuzumab, of 25 g/mL at 10% breakthrough when running at a flow of 10 mL/min in a 0.4 mL HiTrapTM device.
• DBC dynamic binding capacity
• the separation is performed by applying a volume gradient or a pH gradient in step c).
• the separation matrix may be according to the above.
• Figure 1 schematically depicts the HiTrapTM devices (CytivaTM) used for the DBC comparison between prototypes and a commercial product in Example 1.
• Figure 2 shows a chromatogram for Fab and Trastuzumab for a pH gradient slope of 20 mL
• Figure 3 shows a chromatogram for Fab and Trastuzumab for a pH gradient slope of 40 mL
• Figure 4 shows a chromatogram for Fab and Trastuzumab for a pH gradient slope of 60 mL
• Figure 5 shows a chromatogram for Fab and Trastuzumab for a pH gradient slope of 88 mL
• antibody and “immunoglobulin” may be used interchangeably herein and refers to an antigen-binding protein having a basic four-polypeptide chain structure consisting of two heavy (H) chains and two light (L) chains, said chains being stabilized by interchain or intrachain disulfide bonds.
• Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH).
• the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
• Each light chain is comprised of a light chain variable region (VL) and a light chain constant region.
• the light chain constant region is comprised of one domain, CL.
• kappa chain There are two types of light chain in humans, kappa chain and lambda chain.
• the term is to be understood to include any antibody, including but not limited to monoclonal antibodies and bi-specific antibodies, as well as fragments of antibodies, fusion proteins comprising antibodies or antibody fragments and conjugates comprising antibodies or antibody fragments.
• a "kappa light chain-binding polypeptide” and “kappa light chain-binding protein” herein mean a polypeptide or protein respectively, capable of binding to a subclass 1, 3 or 4 kappa light chain of an antibody (also called V K i, V K m and V K iv, as in B H K Nilson et al: J. Biol. Chem. 267, 2234-2239, 1992), and include e.g. Protein L, and any variant, fragment or fusion protein thereof that has maintained said binding property.
• kappa light chain-containing protein is used as a synonym of "immunoglobulin kappa light chain-containing protein” and herein means a protein comprising a subclass 1, 3 or 4 kappa light chain (also called V K i, V K m and V K iv, as in B H K Nilson et al: J. Biol. Chem. 267, 10 2234-2239, 1992) derived from an antibody and includes any intact antibodies, antibody fragments, fusion proteins, conjugates or recombinant proteins containing a subclass 1, 3 or 4 kappa light chain.
• mAb stands for monoclonal antibody
• Fab stands for an antigen binding fragment from an immunoglobulin, comprising a kappa light chain or a lambda light chain.
• bi-specific antibody stands for an antibody that can bind to two different types of antigen or two different epitopes on the same antigen.
• a tri-specific antibody stands for an antibody that can bind to three different types of antigen or three different epitopes on the same antigen.
• DBC means-Dynamic binding capacity and is the binding capacity under operating conditions, i.e., in a packed affinity chromatography column during sample application.
• the DBC of a chromatography resin is the amount of target protein that binds to the resin under given flow conditions before a significant breakthrough of unbound protein occurs. DBC is determined by loading a sample containing a known concentration of the target protein and monitoring the flow-through. The protein will bind to the resin to a certain break point before unbound protein will flow through the column.
• the DBC can be determined on the breakthrough curve at a loss of, for example, 10% protein. This is referred to as the Qbl0% value, or simply Qbl0%.
• Qbl0% value a loss of, for example, 10% protein.
• a sample is applied to a chromatography resin column during a specific residence time and the dynamic binding capacity for each resin is calculated at 10% of the protein breakthrough i.e., the amount of target sample that is loaded onto the column until the concentration of target sample in the column effluent is 10% of the target sample concentration in the liquid sample. If the dynamic binding capacity for each resin is calculated at 80% of the breakthrough capacity, this is referred to as the Qb80% value
• the liquid sample may be referred to as "Clarified Cell Culture Feed” or "CCF".
• a “buffer” is a substance which, by its presence in solution, increases the amount of acid or alkali that must be added to cause unit change in pH.
• a buffered solution resists changes in pH by the action of its acid-base conjugate components.
• Buffered solutions for use with biological reagents are generally capable of maintaining a constant concentration of hydrogen ions such that the pH of the solution is within a physiological range.
• physiological pH refers to the pH of mammalian blood (i.e., 7.38 or about 7.4). Thus, a physiologic pH range is from about 7.2 to 7.6.
• Traditional buffer components include, but are not limited to, organic and inorganic salts, acids and bases.
• Exemplary buffers for use in purification of biological molecules include the zwitterionic or "Good” Buffers, see e.g., Good et al. (1966) Biochemistry 5:467 and Good and Izawa (1972) Methods Enzymol. 24:62.
• Elution liquid or “elution buffer”, which are used interchangeably herein, refers herein to the liquid that is used to dissociate the target substance from the chromatography resin, thereby eluting the binding region-containing protein from the immobilized binding agent, after it has been washed with one or more wash liquids.
• the elution liquid acts to dissociate the target substance without denaturing it irreversibly.
• Typical elution liquids are well known in the chromatography art and may have a different pH (typically lower pH), higher concentrations of salts, free affinity ligands or analogues, or other substances that promote dissociation of the target substance from the chromatography resin.
• Elution conditions refers to process conditions imposed on the target substance-bound chromatography resin that dissociate the target substance from the chromatography resin, such as the contacting of the target substance-bound chromatography resin with an elution liquid or elution buffer to produce such dissociation.
• the elution buffer has a low pH and thereby disrupts interactions between the kappa light chain binding separation matrix and the protein of interest.
• the low pH elution buffer has a pH in the range from about 2 to about 5, most preferably in the range from about 3 to about 4.
• buffers that will control the pH within this range include glycine, phosphate, acetate, and citrate buffers, as well as combinations of these.
• the preferred such buffers are citrate and acetate buffers, most preferably sodium citrate or sodium acetate buffers.
• Cleaning liquid may be an acidic solution or an alkali solution for removing resin residues after elution of the target substance.
• an alkali solution for removing resin residues after elution of the target substance.
• precipitated proteins, hydrophobic proteins, nucleic acids, endotoxins and viruses may be removed by the cleaning liquid.
• alkali solutions are used for the purpose
• Cleaning-in-place is an important process for efficient use of a chromatography column.
• a cleaning procedure that efficiently removes impurities without being harmful to the chromatography resin is required.
• the terms “comprises”, “comprising”, “containing”, “having” and the like can mean “includes”, “including”, and the like; “consisting essentially of” or “consists essentially” is an open- ended term, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
• the inventors had as an objective to invent a separation matrix containing Protein L-derived ligands having an improved stability towards alkaline cleaning procedures, while maintaining a satisfactory efficiency in binding capacity and flow characteristics in a chromatography setting, and preferably better than current commercially available separation matrices.
• the objective has been attained by providing a separation matrix comprising kappa light chain-binding ligands covalently coupled to a porous support, wherein said kappa light chain-binding ligands comprise, consists essentially of, or consists of multimers of alkali-stabilized Finegoldia (former Peptostreptococcus) Protein L domains; and said porous support is a convectionbased chromatography matrix.
• the kappa light chain-binding ligands comprised in the separation matrix of the present invention comprises, consists essentially of, or consists of, multimers of alkali-stabilized Finegoldia Protein L domains.
• the Protein L domains may be any functional Protein L derived domain as long as it is alkali stabilized.
• the Protein L domains are chosen from a functional variant of a Bl domain, a B2 domain, a B3 domain, a B4 domain, a B5 domain, a C2 domain, a C3 domain, a C4 domain and a DI domain, wherein the positions which in an alignment corresponds to positions 10 and 45 in a B2 domain (SEQ ID NO 1) are histidine, and the position which in an alignment corresponds to position 60 in a B2 wt domain (SEQ ID NO 1) is a tyrosine or a glutamine.
• the above-mentioned positions corresponding to positions 10, 45 and 60 in a B2 wt domain (SEQ ID NO 1) are not variable within the functional Protein L domain.
• Protein L domains examples may be:
• SEQ ID NO: 4 (B3: N10H, N45H, N60Y mutations)
• SEQ ID NO:6 (Bl: N10H, N45H, N60Y mutations)
• SEQ ID NO: 8 (B4: N10H, N45H, N60Y mutations)
• SEQ ID NO: 16 (C4: N10H, N45H, N60Y mutations)
• SEQ ID NO: 17 (C4: N10H, N45H, N60Q mutations)
• SEQ ID NO: 18 (DI: N10H, N45H, N60Y mutations)
• SEQ ID NO: 19 (DI: N10H, N45H, N60Q mutations)
• the remaining positions in such a functional Protein L domain may be varied as long as the three- dimensional structure is not altered as compared to that of the B2 wt domain (SEQ ID NO 1), and as long as it at least retains the kappa light chain-binding capacity and is alkali-stabilized as compared to the B2 wt domain (SEQ ID NO 1).
• the variation may be conservative amino acid substitutions for an amino acid with a similar or identical charge, hydrophobicity, etc., and the skilled person is able to determine what such a variation of an amino acid may be.
• the Protein L domain may have at least 90%, 95% or 98% sequence identity, or a 77.5% sequence similarity as determined by BLOSUM matrix of 75, with a gap open penalty of 12, a gap extension penalty of 3, with any one of the amino acid sequences SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18 or SEQ ID NO 19.
• the functional Protein L domain may be a truncated sequence. For instance the positions corresponding to positions 1-4 in B2 wt domain (SEQ ID NO 1) may be deleted. For instance positions corresponding to positions following position 65 in B2 wt domain (SEQ ID NO 1) may be deleted.
• the Protein L domain may have at least 90%, 95% or 98% sequence identity, or a 77.5% sequence similarity as determined by BLOSUM matrix of 75, with a gap open penalty of 12, a gap extension penalty of 3, with any one of the amino acid sequences SEQ ID NQ:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NQ:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:37.
• the kappa light chain-binding ligands comprised in the separation matrix comprise, consists essentially of, or consists of multimers of the alkali-stabilized Protein L domains.
• the multimer may comprise two, three, four, five, six, seven, eight or nine alkali-stabilized Protein L domains.
• the multimers may be a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer or a nonamer.
• the ligands comprise four, five, six or seven alkali-stabilized Protein L domains, such as five or six alkali-stabilized Protein L domains.
• the multimer may further comprise a linker, spacer, or additional amino acid(s).
• the additional amino acid(s) may for instance originate from the cloning process and expression of the ligand or constitute a residue from a cleaved off signalling sequence.
• the skilled person will appreciate and understand that such additional amino acid(s) may vary without impacting the function of the kappa light chain-binding function of the multimer.
• the porous support comprises a convection-based chromatography matrix.
• Said convection-based chromatography matrix may be a fibrous substrate.
• Said fibrous substrate may be based on electrospun polymeric fibres or cellulose fibres, optionally non-woven fibres, which in use form a stationary phase comprising a plurality of pores through which a mobile phase can permeate.
• the fibrous substrate may thus be a fibrous non-woven polymer matrix.
• Such a fibrous substrate can be found in a HiTrap FibroTM unit from CytivaTM.
• Convection-based chromatography support materials enable the combination of high flow rates with high binding capacity and are hence of interest for use in for example mAb purification.
• the polymer fibers may be non-woven fibers. Using a randomly deposited fiber mat (non-woven) structure can encourage impeded flow thereby discouraging channeling.
• the polymer fibers may be electrospun. Electrospinning provides fibers with consistent dimensions and can easily be tuned (for example, by varying atmospheric properties while spinning) to make fibers of different proportions. Electrospinning is a technique that also demonstrates excellent distribution properties, especially useful when creating layered membranes. Fiber mass transfer characteristics have been shown to be similar to those in a monolith structure which allow for flow rate independent separations
• the polymer used in the present invention is not limited to any specific polymer and can be tailored for specific use.
• the polymer may be for example: nylon, poly(acrylic acid), polyacrylonitrile, polystyrene, polysulphone, polyacrylonitrile, polycaprolactone, collagen, chitosan, agarose and polyethylene oxide and combinations thereof.
• the polymer may be derivatised to enhance the solubility and/or other properties of the polymer in order to improve its suitability to be electrospun.
• the derivatised polymer for instance polyether sulfone, cellulose acetate or poly(acrylonitrile-co-acrylic acid) can be treated post electrospinning to regenerate the original polymer or derivatise further to create a new functionality.
• the polymer used in the invention is typically cellulose. Cellulose is often used as it is readily available, cheap, biodegradable, biologically compatible and has a hydrophilic surface resulting in low non-specific binding.
• the fibres may have a diameter of lOnm to lOOOnm.
• the fibres may have a diameter of 200nm to 800nm and may even have a diameter of 300nm to 400nm. Fibres of this size yield improved consistency of pore size and size distribution.
• the fibres may have a mean length of greater then 10cm. Fibres generated by electrospinning are typically much longer than the fibres found in conventional chromatography media. Longer fibres deliver improved layering properties. In some cases, where the electrospinning comprises a fibre emanating from a single source, a single continuous fibre may be produced and the membrane formed from this fibre alone, or from a small number (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) of long fibres.
• the pores of the stationary phase may be lOnm to 10pm in diameter, often 25nm to 5pm and can be 50nm to 2 pm in diameter.
• Use of pore sizes within these size ranges can help to minimise fouling of the chromatography medium and decrease product loss due to polarization, concentration and rejection at particle interfaces. However, the pores remain small enough to minimise the loss of target components passing through the membrane without coming into contact with the medium.
• the selection of these pore sizes ensures good utilisation of capacity and sharper breakthrough curves. It has previously been demonstrated that for a membrane structure with similar pore size the fibre structures were 1.5 - 2 times more permeable to aqueous flow than a traditional membrane produced by phase inversion. This is due to the relatively high surface porosity that the electrospinning process yields.
• a non-woven fibre membrane with average fibre diameter of 300nm contained an average pore size of ca. 500nm, yet yielding a porosity of 49%.
• electrospinning fibre membranes it is possible to achieve a high level of surface porosity of high distribution well above what could be expected from traditionally formed membranes as in their case the relationship of decreasing surface porosity with decreasing pore size is dominant.
• the pores may have a narrow size distribution, wherein the standard deviation in pore diameter is preferably less than or equal to 250nm. Pore size uniformity is one of several factors as well as, axial and radial diffusion and sorption kinetics, that has been shown to have an impact on key performance factors in chromatography (particularly affinity chromatography) such as breakthrough curve (BTC) sharpness.
• the fibers as disclosed above are functionalized before the ligand is immobilized thereto.
• the preparation of a convective based chromatography matrix is disclosed in for instance WO/2019/137869 and WO/2013/068741, which are incorporated by reference in their entirety.
• membrane is often used interchangeably with the porous support. It should be clear that whenever “membrane” is used, it refers to the porous support material as disclosed above.
• the ligand density achieved on the porous support is at least 20 mg ligand /ml porous support, or at least 25 mg ligand/ml porous support, such as 30 mg ligand /ml porous support, such as at least 35 mg ligand/ml porous support, or at least 40 mg ligand/ml porous support, or at least 45 mg ligand/ml porous support, or at least 50 mg/ml porous support.
• the separation matrix of the present has a dynamic binding capacity (DBC) of a kappa light chaincomprising antibody, such as Trastuzumab, of 25 g/mL at 10% breakthrough when running at a flow of 10 mL/min in a 0.4 mL HiTrapTM device.
• the separation matrix of the present has a dynamic binding capacity (DBC) of a kapa light chain-comprising antibody, such as Trastuzumab, of 45 g/mL at 10% breakthrough when running at a flow of 10 mL/min in a 0.4 mL HiTrapTM device.
• the present invention also relates to methods for isolation of a kappa light chain-binding protein. Said method is preferably performed with the separation matrix as disclosed above.
• the isolation may be from a cell culture or other liquid comprising kappa light-chain binding proteins.
• the present disclosure provides for a method of isolating a kappa light chain-containing protein comprising the steps of: a) contacting a liquid sample comprising a kappa light chain-containing protein with a separation matrix; b) washing said separation matrix with one or a combination of several washing liquids; c) eluting the kappa light chain-containing protein from the separation matrix with an elution liquid; and d) cleaning the separation matrix with a cleaning liquid.
• the separation matrix has a dynamic binding capacity (DBC) of the kappa light chain-containing protein, such as Trastuzumab, of 28 g/mL at 10% breakthrough when running at a flow of 10 mL/min in a 0.4 mL HiTrapTM device .
• DBC dynamic binding capacity
• the separation matrix as disclosed above is used in said method.
• variable domains therein VL; VH
• VL variable domains therein
• the pairing of two different light and two different heavy chains may lead to a large number of mispairing, as normally only one specific asymmetric combination is wanted, and a multitude of combinations achieved will be nonfunctional or unwanted molecules, such as for instance monospecific homodimers.
• a method for separation of bispecific antibodies comprising the steps of: a) contacting a liquid sample comprising kappa light chain-containing proteins with a separation matrix, b) washing said separation matrix with one or a combination of several washing liquids, c) eluting the kappa light chain-containing protein from the separation matrix with an elution liquid and d) cleaning the separation matrix with a cleaning liquid.
• the separation matrix has a dynamic binding capacity (DBC) of the kappa light chain-containing protein, such as Trastuzumab, of 28 g/mL at 10% breakthrough when running at a flow of 10 mL/min in a 0.4 mL HiTrapTM device.
• DBC dynamic binding capacity
• the separation matrix as disclosed above is used in said method.
• the elution step may be performed with a volume gradient. This is shown in Example X.
• the elution step may be performed with a pH gradient.
• This method enables to separate bi-specific antibodies from monospecific antibodies, or mismatched antibodies, based on the presence of a kappa light chain.
• a monoclonal antibody has two identical kappa light chain.
• a bispecific antibody can be designed to comprise two different kappa light chain.
• Any antibody not comprising a kappa light chain such as two lambda light chains, will not bind the separation matrix and consequently be present in the effluent flow during step a) or be washed away during step b). Any antibodies that have at least one kappa light chain will bind to the separation matrix. Upon elution, antibodies that have only one kappa light chain will elute prior to antibodies that have two kappa light chains.
• step 4 Immobilization of kappa light chain-binding ligands on to the material obtained in step 3 comprising;
• the desalting was performed by using Pre-packed PD10 columns containing Sephadex-G-25 medium.
• the columns were equilibrated with 4 column volumes of de-gassed desalting solution (0.15 NaCI, 1 mM EDTA) prior to loading the protein (max 2.5 mL). The eluted fractions were collected and combined. The desalted solution was diluted 20 times by desalting solution and the absorbance at 276 nm was measured and corrected by a blank of desalting buffer. By using the determined protein concentration, it was then possible to calculate the desired amount of desalted protein solution to be used during immobilization.
• a separation matrix according to the present invention was prepared as above, and the Dynamic binding capacity was analysed.
• A £ L c
• A is the UV absorption at 280 nm
• E is the absorptivity coefficient (for polyclonal IgG E is 1.38 ml/(mg x cm).
• L is the path length of the cell holder, c is the concentration of the solution.
• 3.4 mL of purified Trastuzumab (30 g/L) was diluted with 20 mM Phosphate 150 mM NaCI pH 7.2 in a total volume of 200 mL. The concentration was determined by UV measurements at 280 nm using 96 well UV plate, 200 pL/well. Blank was 20 mM Phosphate 150 mM NaCI pH 7.2. The Trastuzumab concentration was calculated using the extinction coefficient 1.48.
• dAB is an domain antibody comprising the variable light chain and was produced according to the method described in the article "Recombinant production of a V L single domain antibody in Escherichia coli and analysis of its interaction with Peptostreptococcal protein L " (Protein Expression and Purification, Volume 51, Issue 2, February 2007, Pages 253-259). 3.45 mL of dAb (14.5 g/L) was diluted with 20 mM Phosphate 150 mM NaCI pH 7.2 in a total volume of
• the concentration was determined by UV measurements at 280 nm using 96 well UV plate, 200 pL/well. Blank was 20 mM Phosphate 150 mM NaCI pH 7.2. The dAb concentration was calculated using the extinction coefficient 1.6.
• the ligands were coupled to the porous support as disclosed in Example 6 in WO/2019/137869 and WO/2013/068741.
• a membrane single disc of the porous support coupled with ligand was installed in a device and connected to a chromatographic system, AKTAavant 25 IP31154, 10 mm cell.
• the membrane was equilibrated with the equilibration/binding buffer before protein sample load with gammanorm or Trastuzumab, respectively.
• the capacity at 10 % breakthrough (Qbl0%) was calculate and reported.
• the flow used during equilibration and washing of the membrane was 20 mL/minutes. The flow was reduced during protein load to 10 mL/min.
• the analysis was performed on duplicate membrane discs and each disc was analyzed two times. The first run was a blank without protein load followed by two frontal analysis runs with protein.
• Asub absorbance contribution from non-binding mAb
• A(V) absorbance at a given applied volume
• V app volume applied until 10% breakthrough
• V S ys system dead volume
• Co liquid sample concentration
• the sample solution comprised 0,5 mg protein.
• the DBC decreases between run 1 and 2 and increases again for run 3 then drops for run 4 again. This is due to the fact that no blank run was done between the analyses 1 and 2 and between 3 and 4, respectively.
• Table3 Dynamic Binding Capacity Trastuzumab Results In this experiment a blank run was added between the two runs 1 and 2. This shows that a blank run should be performed between each run.
• test 2 may be attributed to the fact that a Cl P with 0.1 M NaOH has been performed on the material before the test 2, whereas test 1 is performed without a preceding CIP wash.
• the DBC of prototypes according to the present invention were compared to a commercial product with the same convection-based matrix.
• the matrix was comprised in a HiTrapTM Device as shown in Fig. 1.
• the HiTrapTM prototype devices (1) have a volume of 0.4 mL.
• the device (1) contains two stacked Fibro prototype membranes (2) with non-woven material layers (3) in between as filling material.
• the disc diameter is 26.4 mm.
• Prototype 1 comprised a separation matrix with 52 mg ligand/ml membrane.
• Prototype 2 comprised a separation matrix with 34 mg ligand/ml membrane.
• Table 6 DBC comparison with commercial product and protypes in a HiTrapTM device.
• the prototypes exhibit at least as good a DBC as the commercial product FibroTM PrismA, both for Qb5 and QblO.
• the Fab fragment was produced from Trastuzumab by papain cleavage.
• the Trastuzumab solution was adjusted to pH 7.4 by addition of 0.5 M Sodium phosphate and then diluted 1+1 in digestion buffer (25 mM Na-phosphate, 1 mM EDTA, 5 mM mercapto-ethanol, pH 7.5). Final volume was approx. 100 mL Papain crystals were added to the solution. The solution was incubated at 37°C over-night. Thereafter, Antipain (papain inhibitor) was added to the digested Trastuzumab.

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